The goal of the proposed research is to identify and characterize the proteins involved in DNA replication in mammalian cells. We are approaching this problem by analyzing the cellular protein requirements for replication of the SV40 genome in vitro. During the past grant period we developed methods for the fractionation of the crude SV40 DNA replication system and demonstrated that multiple cellular factors are required to reconstitute efficient DNA synthesis in vitro. A number of the required proteins were purified to near homogeneity and partially characterized. During the next grant period we plan to complete the identification of the components of the mammalian replication machinery and to reconstitute the complete replication reaction with purified proteins. We also plan to analyze the functional roles of the individual cellular replication proteins with particular emphasis on the proteins involved in the initiation reaction.